The TaqMan probe principle relies on the 5´–3´ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection.[2] As in other quantitative PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however, the TaqMan probe significantly increases the specificity of the detection. TaqMan probes were named after the videogame PacMan (Taq Polymerase + PacMan = TaqMan) as its mechanism is based on the PacMan principle.[3]

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TaqMan probes consist of a fluorophore covalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3’-end[4] (Figure 1). Several different fluorophores (e.g. 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescein, acronym: TET) and quenchers (e.g. tetramethylrhodamine, acronym: TAMRA) are available.[5] The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source via FRET (Fluorescence Resonance Energy Transfer).[6] As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals (Figure 1).

TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers. (Unlike the diagram, the probe binds to single stranded DNA.) As the Taq polymerase extends the primer and synthesizes the nascent strand (again, on a single-strand template, but in the direction opposite to that shown in the diagram, i.e. from 3' to 5' of the complementary strand), the 5' to 3' exonuclease activity of the Taq polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the quantitative PCRthermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.